Phycobilisomes are complexes of phycobiliproteins and colorless polypeptides which function as the major light harvesting antennae in blue-green and red algae (Gantt, (1975) xe2x80x9cPhycobilisomes: light harvesting pigment complexes,xe2x80x9d BioScience 25:781-788). Naturally-occurring phycobilisomes from different organisms share a number of common properties, including: (1) extremely high xe2x80x9ccomplex molecular weightsxe2x80x9d (5-20xc3x97106 daltons) i.e., the weight of one mole of a phycobilisome complex comprised of multiple molecules; (2) multiple absorption maxima in the visible range of the electromagnetic spectrum; (3) high molar absorptivities (emax greater than 107 Mxe2x88x921xc2x7cmxe2x88x921); (4) efficient ( greater than 90%) directional vibrational energy transfer among constituent phycobiliproteins, commonly from one or more sensitizing species to a terminal acceptor capable of fluorescence; (5) large Stokes shifts relative to isolated phycobiliproteins; (6) high quantum yields of constituent phycobiliproteins; (7) high solubility in aqueous buffers; (8) allophycocyanin-containing core structures; and (9) precisely defined phycobiliprotein and linker polypeptide composition and supramolecular organization.
Morphologically, phycobilisomes are complex assemblies of oligomeric phycobiliprotein discs arranged in ordered stacks referred to as xe2x80x9crodsxe2x80x9d. In general, several arm-like rods radiate out from a core assembly, also comprised of rods. Phycobilisomes from different organisms are morphologically and stoichiometrically diverse, having different numbers and types of constituent phycobiliproteins and rods. In general, peripheral rods are comprised of phycoerythrocyanin, phycoerythrin, and/or phycocyanin and associated linker proteins, and the core is comprised of allophycocyanin and associated linker proteins. The colorless polypeptides are involved in the assembly and positioning of the phycobiliproteins within the phycobilisomes for proper stability and energy transfer. The major criterion for the functional integrity of these complexes is the demonstration that they exhibit highly efficient transfer of energy between component phycobiliproteins, for example, in Porphyridium cruentum phycobilisomes from phycoerythrin (PE) to phycocyanin (PC) and finally to allophycocyanin (APC).
Supramolecular complexes comprising phycobilisomes are well-known in the art, as evidenced by the substantial body of literature on preparative methods (e.g., Gantt, E. 1986, xe2x80x9cPhycobilisomes. In: Photosynthesis III: Photosynthetic Membranes and Light Harvesting Systemsxe2x80x9d (L. A. Staehelin and C. J, Arntzen, eds.), pp.260-268, Springer-Verlag, NY; Grossman, A. R. et al. 1993, xe2x80x9cThe phycobilisome, a light-harvesting complex responsive to environmental conditions,xe2x80x9d Microbiological Reviews 57:725-749; Hiller, et al., 1982, xe2x80x9cIsolation of intact detergent-free phycobilisomes by trypsin.xe2x80x9d FEBS Lett. 156:180-184), rod and core subassemblies (e.g., Lundell, et al. 1983a, xe2x80x9cMolecular architecture of a light-harvesting antenna: core substructure in Synechococcus 6301 phycobilisomes: two new allophycocyanin and allophycocyanin B complexes,xe2x80x9d J. Biol. Chem., 258:902-908; Lundell, et al., 1983b, xe2x80x9cMolecular architecture of a light-harvesting antenna: quaternary interactions in the Synechococcus 6301 phycobilisome core as revealed by partial tryptic digestion and circular dichroism studies,xe2x80x9d J. Biol. Chem., 258:8708-8713; Lundell, et al., 1983c, xe2x80x9cMolecular architecture of a light-harvesting antenna: structure of the 18S core-rod subassembly of the Synechococcus 6301 phycobilisome,xe2x80x9d J. Biol. Chem., 258:894-901; Glazer, A. N. 1985a, xe2x80x9cLight harvesting by phycobilisomes,xe2x80x9d Annual Rev. Biophys. and Biophys. Chem., 14:47-77), phycobilisome-photosystem complexes (e.g., Diner, B. A. 1979, xe2x80x9cEnergy transfer from phycobilisomes to photosystem II reaction centers in wild type Cyanidium caldarium,xe2x80x9d Plant Physiol., 63:30-34; Gantt E, et al. (1988), xe2x80x9cPhotosystem II-phycobilisome complex preparations,xe2x80x9d Meth. Enzymol. 167, 286-290; Clement-Metral, J. D. and Gantt (1983a), xe2x80x9cIsolation of oxygen-evolving phycobilisome-photosystem II particles from Porphyridium cruentum,xe2x80x9d FEBS Letters 156:185-188; Clement-Metral J D, et al. (1983b), xe2x80x9cA photosystem II-phycobilisome preparation from the red alga Porphyridium cruentum: oxygen evolution, ultrastructure, and polypeptide resolution,xe2x80x9d Arch. Biochem. Biophys. 238:10-17; Kirilovsky D, et al. (1986). xe2x80x9cFunctional assembly in vitro of phycobilisomes with isolated photosystem II particles of eukaryotic chloroplasts,xe2x80x9d J. Biol. Chem., 261:12317-12323), phycobilisome-membrane preparations (e.g., Clement-Metral, J. D., et al. (1971), xe2x80x9cFluorescence transfer in glutaraldehyde fixed particles of the red alga Porphyridium cruentrum (N),xe2x80x9d FEBS Letters 12:225-228), phycobilisome dissociation (e.g., Rigbi, et al. (1980), xe2x80x9cCyanobacterial phycobilisomes: Selective dissociation monitored by fluorescence and circular dichroism.xe2x80x9d Proc. Natl. Acad. Sci. USA, 77:1961-1965) and reconstitution (e.g., Gantt, et al. (1979), xe2x80x9cPhycobilisomes from blue-green and red algae: Isolation criteria and dissociation characteristics,xe2x80x9d Plant Physiology 63:615-620; Kirilovsky et al. (1986), Glick, et al. (1983), xe2x80x9cRole of the colorless polypeptides in phycobilisome reconstitution from separated pycobiliproteins,xe2x80x9d Plant Physiol., 69:991-997), genetic modifications (e.g., Bryant, D. A., 1991, xe2x80x9cCyanobacterial phycobilisomes: progress toward complete structural and functional analysis via molecular genetics,xe2x80x9d In L. Bogorad and I.K. Vasil (ed.), Cell Culture and Somatic Genetics of Plants. Molecular Biology of Plastids and Mitochondria, Vol. 7, pp. 257-300, Academic Press, San Diego, Calif.); Yamanaka, et al. (1978), xe2x80x9cCyanobacterial phycobilisomes. Characterization of the phycobilisomes of Synechococcus sp. 6302,xe2x80x9d J. Biol. Chem., 253:8303-8310; Yamanaka, et al. (1980), xe2x80x9cMolecular architecture of a light-harvesting antenna. Comparison of wild type and mutant Synechococcus 6301 phycobilisomes,xe2x80x9d J. Biol. Chem., 255:11004-11010), and environmental effects (e.g., Grossman et al. (1993)), including chromatic adaptation (e.g., Bryant, et al. 1981, xe2x80x9cEffects of chromatic illumination on cyanobacterial phycobilisomes: Evidence for the specific induction of a second pair of phycocyanin subunits in Pseudanabaena 7409 grown in red light,xe2x80x9d Eur. J. Biochem. 119:415-424).
Isolated phycobilisomes readily dissociate into free phycobiliproteins and a variety of phycobiliprotein complexes under all but the most favorable conditions. Low to moderate ionic strength ( less than 0.5 M phosphate), low phycobilisome concentration ( less than 1 mg/ml), and low and high temperatures lead to dissociation of phycobilisomes (Katoh, (1988) Methods in Enzymology, 162:313-318; Gantt et al., (1979)). Freezing of algae is also reported to lead to destruction of phycobilisomes (Gantt et al., (1972) Journal of Cell Biology, 54:313-324).
Isolated phycobiliproteins, the component fluorescent proteins of phycobilisomes, have been used as labels in immunoassays. See e.g., Stryer et al., U.S. Pat. No. 4,520,110 and Kronick et al. (1983) Clinical Chemistry, 29:1582-1586. However, because of the difficulty in isolating and manipulating intact phycobilisomes, the art has not recognized that these macromolecular assemblies could be similarly utilized. Because the signal which phycobilisomes can provide is theoretically so much larger than that of isolated phycobiliproteins, there is a need in the art for methods of treating phycobilisomes so that they can be used as detectable markers for a host of assays and other applications.
One object of this invention is to provide supramolecular complexes with diverse spectral properties for use as highly detectable tracers and labels.
Another object of this invention is to provide a versatile set of highly sensitive signal-generating systems and conjugates for use in, inter alia, various assay methods.
Yet another object of this invention is to provide biotransducers comprising phycobilisomes or phycobilisome complexes immobilized on a manufactured solid support.
Still another object of this invention is to provide methods for performing specific binding assays using signal-generating systems comprising phycobilisomes as detectable labels. These and other intentions of this invention are achieved by one or more of the following embodiments.
In one embodiment, this invention provides an isolated, soluble, stabilized phycobilisome comprising two or more phycobiliproteins specifically connected by at least one linker polypeptide. The stabilized phycobilisome of this embodiment may comprise at least one peripheral rod, or a core complex and no peripheral rods, or a core complex and at least one disc. In one mode, the stabilized phycobilisome of this embodiment comprises an anchor polypeptide. In a particular mode, the different proteins making up the stabilized phycobilisome of this embodiment are not all found in a single algal strain, but rather are proteins whose sequences are encoded by more than one distinct algal strain; in other words, some of the proteins making up the phycobilisomes of this mode may originally derive from an algal strain different from the strain that is the source of other proteins in the phycobilisome. In another particular mode, the stabilized phycobilisome is reconstituted from a mixture containing phycobilisome components, which may include isolated phycobiliproteins and/or isolated linker polypeptides, and optionally partially reconstituted phycobilisomes. In a preferred mode of this embodiment, the stabilized phycobilisome is modified by covalent attachment of desired chemical moieties, the chemical moieties optionally being attached to a particular portion of the phycobilisome. In yet another mode, the isolated, stabilized phycobilisome of this embodiment is functionally coupled to another signal-generating system.
In another embodiment, this invention provides a phycobilisome conjugate comprising a phycobilisome conjugated to a molecular species selected from the group consisting of ligands, receptors, and signal-generating molecules, where the phycobilisome comprises a plurality of phycobiliproteins specifically connected by at least one linker polypeptide, the molecular species preferably being attached to a single type of phycobiliprotein or a single type of linker polypeptide or an anchor peptide. Alternatively, the molecular species may be attached to a particular portion of the phycobilisome. In one mode of this embodiment, the phycobilisome comprises at least one protein encoded by each of at least two different algal strains. In an alternative mode of this embodiment, the phycobilisome is reconstituted from a mixture containing isolated phycobiliproteins, isolated linker proteins, or a mixture thereof In yet another mode of this embodiment, the phycobilisome is functionally coupled to a signal-generating system. The molecular species conjugated to the phycobilisome may be, for example, streptavidin, avidin, an antibody, biotin, a drug, an antigen, a hapten, a nucleic acid, a carbohydrate, or a lectin.
In yet another embodiment, this invention provides an isolated, functionally intact phycobilisome comprising a plurality of phycobiliproteins specifically connected by at least one linker polypeptide, where the phycobilisome is immobilized on a solid support. In a preferred mode of this embodiment, the immobilized phycobilisome is stabilized. In another preferred mode, the immobilized phycobilisome is covalently attached to a molecular species selected from the group consisting of ligands, receptors, and signal-generating molecules, and more preferably, the molecular species is attached to one type of constituent phycobilisome protein or to a particular portion of the phycobilisome. In one mode of this embodiment, the immobilized phycobilisome comprises at least one protein encoded by each of at least two different algal strains. In an alternative mode of this embodiment, the immobilized phycobilisome is reconstituted from a mixture containing isolated phycobiliproteins, isolated linker proteins, or a mixture thereof. In yet another mode of this embodiment, the immobilized phycobilisome is functionally coupled to another signal-generating system. Alternatively, the immobilized phycobilisome may be a functional component of a biotransducer. The solid support may be selected from the group consisting of a synthetic membrane, a polymer, a microparticle, silicon, and glass. In a particular mode of this embodiment, the invention provides a manufactured solid support containing a plurality of immobilized phycobilisomes, where the phycobilisomes are immobilized on the solid support in a structurally ordered arrangement thereby forming a pattern on the solid support. In an alternative mode of this embodiment, the invention provides a manufactured solid support containing a plurality of immobilized phycobilisomes, where the phycobilisomes are all immobilized on the solid support in the same orientation with respect to the solid support.
In still another embodiment, this invention provides an input system for a transducer comprising conversion means for receiving ultraviolet or visible light and directionally transferring light energy of this light; and coupling means for receiving the directionally transferred light energy and delivering the light energy to a transducer. Preferably, the coupling means comprises an optical fiber or a waveguide; preferably, the conversion means comprises a phycobilisome.
In yet another embodiment, this invention provides an environmentally responsive optical sensor comprising conversion means for receiving ultraviolet or visible light and directionally transferring light energy of this light, such that transfer of light energy is dependent on an environmental condition; and sensor means for receiving the directionally transferred light energy and producing an indication of the environmental condition. In particular, one or more environmental conditions may affect a characteristic of the transferred light energy, such as the energy level of transferred light energy. In a preferred mode of this embodiment, the directionally transferred light energy comprises a photon of a particular energy level, the energy level being dependent upon the environmental condition. More preferably, the conversion means comprises a phycobilisome.
In still another embodiment, this invention provides a system for processing a light signal comprising conversion means for receiving ultraviolet or visible light and directionally transferring light energy of this light; and processing means for receiving and processing the directionally transferred light energy. In a preferred mode of this embodiment, the processing means comprises an optical fiber operative to transmit the light signal and/or a photosensor. Preferably, the directionally transferred light energy comprises a photon, and/or the conversion means comprises a phycobilisome.
In yet another embodiment, this invention provides a method for performing a specific binding assay comprising contacting a sample comprising an analyte with a specific binding partner; determining the amount of the analyte present in the sample by means of its ability to specifically bind to the specific binding partner, where a component of the assay is detectably labeled with a signal-generating system comprising phycobilisomes, the phycobilisomes being self-assembling complexes of phycobiliproteins and linker proteins, where each phycobilisome comprises at least one rod. The detectably-labeled assay component is selected from the group consisting of a specific binding partner of the analyte, reagent molecules having the same chemical identity as the analyte, and reagent molecules which compete with the analyte for specific binding to the specific binding partner. Typically, the competitive reagent molecules will have the same binding specificity as the analyte, and optionally they may have similar affinity for the specific binding partner. In a preferred mode of this embodiment, the analyte or its specific binding partner is attached to a solid phase. The solid phase may be, for example, a synthetic membrane, a polymer, a microparticle, silicon, or glass, and the analyte may be, for example, a nucleic acid, a drug, a ligand, an antigen, a hapten, an antibody, or a carbohydrate.
These and other embodiments of the invention provide the art with an extremely sensitive, nonisotopic detection means for assaying analytes and for sensing molecular events and environmental conditions. Unlike enzymatic labels, phycobilisomes can be quantitatively detected without accessory substrates, chromogens, cofactors, or timed incubations. Alternatively, phycobilisomes can be functionally coupled to enzymes or other signal-generating molecules to amplify or transduce molecular events, thereby generating a more preferred assay signal or transducer output.
Phycobilisomes provide labels of high sensitivity due inter alia to their extremely large molecular weights, extinction coefficients, and energy transfer efficiencies, as well as to the high quantum yields of constituent phycobiliproteins. Directional energy transfer within phycobilisomes occurs from one or more xe2x80x9csensitizing speciesxe2x80x9d to a terminal acceptor. A sensitizing species is a first fluorophor having an emission peak capable of exciting a second (xe2x80x9cacceptorxe2x80x9d or xe2x80x9cemitterxe2x80x9d) fluorophor. Such energy transfer has application in homogeneous specific binding assays and in transducers comprising immobilized phycobilisomes.
It is a discovery of the present invention that phycobilisomes can be stabilized, conjugated, and/or modified so that they can be used intact in a variety of assays and formats. Among other things, this invention provides homogeneous preparations of isolated, soluble, stabilized phycobilisomes. Phycobilisomes may be isolated from the producing organisms after being stabilized in situ prior to cell disruption or in membrane-bound form following cell disruption. Alternatively, phycobilisomes may be isolated intact prior to in vitro stabilization or conjugation or immobilization. In yet another mode of operation, phycobiliproteins and linker proteins can be isolated and reconstituted in vitro to form phycobilisomes.
Phycobilisomes and phycobilisome complexes of the invention may be structurally stabilized to ensure that constituent phycobiliproteins, linker polypeptides and specifically bound components remain physically attached to one another throughout preparation and use. For applications requiring intra-phycobilisome energy transfer among constituent phycobiliproteins, this invention provides internally coupled phycobilisomes, prepared by stabilization methods that preserve inter-subunit energy transfer. Such internally coupled phycobilisomes may be detected by fluorescent, optoelectronic, piezoelectric, photometric, spectroscopic or visual means, among others. Structurally stabilized phycobilisomes which are not internally coupled are still useful as labels in specific binding assays, even though they do not provide the large Stokes shift, directional energy transfer or extraordinary fluorescence intensity of internally coupled phycobilisomes. Uncoupled phycobilisomes are primarily used as labels in fluorescent, photometric, spectroscopic, piezoelectric or visual-read assays or, alternatively, as high molecular weight scaffolds for attachment of signal-generating molecules (e.g., enzymes, fluorophores, luminescent or electroactive compounds) that can be detected by alternative (e.g., enzymatic, fluorescent, luminescent, optoelectric or electrochemical) means.
Definitions
The term xe2x80x9cphycobilisomexe2x80x9d as used herein means a supramolecular light-absorbing structure comprising at least one phycobiliprotein-containing rod and includes phycobilisomes; phycobilisome subassemblies; rod or core fractions; uncoupled, functionally altered or damaged phycobilisomes; genetically, physically, environmentally or chemically modified phycobilisomes; chromatically adapted phycobilisomes; isolated or partially isolated phycobilisomes; dissociated or partially dissociated phycobilisomes; reconstituted or rearranged or recombinant or hybrid phycobilisomes. Phycobilisomes as contemplated by the present invention contain two or more phycobiliproteins specifically connected by one or more linker polypeptides, where the two or more phycobiliproteins are in a particular orientation dictated by the linker polypeptide, with the orientation typically facilitating energy transfer between the phycobiliproteins. The xe2x80x9clinker polypeptidesxe2x80x9d affect the phycobilisomes in a number of ways. First, phycobilisome linker polypeptides can determine the aggregation state and geometry of the particular biliproteins with which they interact. Second, they modulate the spectroscopic properties of the biliprotein. Third, they determine the location of the biliprotein within the phycobilisome and bridge between the biliprotein subcomplexes within the intact structure. In other words, linker polypeptides are proteins which bind two phycobiliproteins specifically and, upon binding, orient the phycobiliprotiens to enhance energy transfer between them. Linker polypeptides also dictate the defined and reproduce able supramolecular composition of phycobilisomes, as distinct, for example, from chemically cross-linked fluorescent polymers of fluorescent proteins. In addition, a large linker polypeptide may participate both in the assembly of the phycobilisome and in the attachment of the phycobilisome to the photosynthetic membrane.
Under appropriate conditions (e.g., high ionic strength), a mixture of isolated phycobiliproteins and linker polypeptides will form supramolecular structures analogous to those of naturally-occurring photosynthetic systems. Such complex structures form as a result of self-assembly directed by the linker polypeptides. Different linker polypeptides determine the composition of the phycobilisome, allowing self-assembly of multiple hexamers into a phycobilisome via the linker polypeptides. Typical structures created by this self-assembly will be one or more peripheral rods, or a phycobilisome core complex, or a complex of phycobiliproteins and core complex, connected by linker polypeptide(s). These self-assembled structures, or molecular entities having the same structure obtained by other means, are phycobilisomes as contemplated herein.
As used herein, the term xe2x80x9crodxe2x80x9d means a peripheral rod or core complex or disc-and-core-complex or combination or subassembly having at least two discs joined by at least one linker polypeptide or at least one disc joined to a core complex by at least one linker polypeptide. Rods are known in the art to be stacks of multimeric phycobiliprotein discs joined by linker polypeptides (e.g., Glazer (1985a)). The term xe2x80x9cdiscxe2x80x9d as used herein means a multimeric phycobiliprotein assemblage that can be reconstituted in vitro from isolated phycobiliprotein subunits (Grossman et al. (1993)). Such discs are typically either trimeric (single disc) or hexameric (double disc) and can be interconverted in vitro under suitable conditions (e.g., Glazer et al. (1971)). The core complex of a phycobilisome typically comprises at least two hexameric discs and associated linker polypeptides. Phycobilisomes of the instant invention, comprising at least one rod which comprises at least two discs, preferably share the common property of directional energy transfer, also referred to herein as xe2x80x9csidedness.xe2x80x9d
Phycobilisomes of the instant invention can be distinguished from isolated phycobiliproteins on structural criteria. Structurally, a phycobilisome comprises at least two hexamers (3 alpha and 3 beta subunits each) of a phycobiliprotein or phycobiliproteins joined by at least one linker polypeptide. While the phycobiliproteins R-PE and B-PE contain a gamma subunit (denoted a xe2x80x9clinkerxe2x80x9d by some authors), isolated R-PE and B-PE are each distinguishable from a phycobilisome in that they are not connected to a second hexamer through a linker polypeptide. However, when two R-PE or two B-PE hexamers are linked by the gamma subunit in the orientation found between these two phycobiliporteins in nature, the resulting rod-like supramolecular entity may be considered a phycobilisome as the term is used herein. Phycobilsomes as contemplated by this invention are also distinct from compositionally and architecturally heterogeneous conjugates that have been prepared by covalently cross-linking isolated phycobiliproteins and other polypeptides.
While most rods in native phycobilisomes are stacks of three or four phycobiliprotein hexameric discs, this invention also contemplates rods with as few as two discs connected by linker polypeptide(s). The phycobilisome core complex is made up of the phycobiliprotein allophycocyanin (APC). Phycobiliproteins of the core form rods containing APC which contain other linker(s) and/or modified subunits. Examples of possible phycobilisomes, as defined under the instant invention, are APC linked to APC via linker polypeptides, APC linked to PC via linker polypeptides and PE linked to PC via linker polypeptides. Larger complexes containing additional phycobiliproteins remain within the definition of phycobilisomes, so long as the complex contains at least two phycobiliproteins which are linked and positioned in the complex by a linker polypeptide. Preferably, phycobilisomes of this invention will have at least three phycobiliprotein hexamers, and more preferably, the phycobilisomes of this invention will have at least the number of phycobiliproteins found in a single rod in nature or in the core complex.
xe2x80x9cStabilized phycobilisomesxe2x80x9d are stable even under conditions of dilute ionic strength ( less than 0.5 M) and protein concentration ( less than 1 mg/ml), in contrast with native phycobilisomes. In addition, they are stable in the presence of glycerol, sucrose, and polyethylene glycol. Typically, the phycobilisomes are stabilized by means of a gentle crosslinking treatment, such as with formaldehyde or very low concentrations of glutaraldehyde. Other medium-, short- or zero-length crosslinking reagents may also be used.
Stabilized phycobilisomes that resist dissociation in dilute solution ( less than 1 mg/ml protein) and low ionic strength buffers ( less than 0.5 M) but are not energetically coupled (i.e., do not exhibit intra-phycobilisome energy transfer) may be used for embodiments of the invention which do not require internal energy transfer (i.e., internal coupling) between constituent phycobiliproteins, such as fluorescent, photometric or visual-read specific binding assays relying on the high molar extinction coefficient of phycobilisome labels. Excitation of the sensitizing phycobiliproteins of a structurally stabilized, energetically uncoupled phycobilisome does not lead to a major emission peak by the terminal acceptor. Stabilized phycobilisomes which retain the property of inter-phycobiliprotein energy transfer, by contrast, are referred to as xe2x80x9cinternally coupled phycobilisomesxe2x80x9d and considered to be xe2x80x9cfunctionally intact.xe2x80x9d Phycobilisomes which are functionally intact have a major emission peak at the wavelength of the terminal acceptor.
It will be understood to those of skill in the art that the extremely effective and efficient light-harvesting properties of internally coupled phycobilisomes provide distinct advantages for applications requiring highly sensitive detection and/or efficient signal transduction. It will also be apparent to the skilled artisan from the instant disclosure that structurally stabilized phycobilisomes which are internally uncoupled are suitable for certain applications described herein (e.g., Assay Example 1 (photometric immunoassay); Assay Example 5 (visual microliter immunoassay); and Assay Example 6 (immunochromatographic dipstick)). Other applications involving energy transduction and/or biotransducers (e.g., Energy Transduction Examples 1 and 2), may use either phycobilisomes that are internally coupled or, alternatively, structurally stabilized, internally uncoupled phycobilisomes which are conjugated to other molecular species (e.g., signal-generating molecules.)
xe2x80x9cIsolated phycobilisomesxe2x80x9d according to this invention are phycobilisomes that are not complexed to an intact thylakoid membrane. Phycobilisomes may be solubilized from thylakoid membranes by treatment with surfactants, detergents, lipids, phospholipids and other amphipathic molecules well-known in the art. Isolated phycobilisomes may be complexed to photosystem complexes which may in turn contain a thylakoid fragment, but when the isolated phycobilisomes of this invention are complexed to membrane structures, the membrane structures differ from naturally occurring thylakoid membranes in at least one characteristic. Typically a membrane-bound isolated phycobilisome will be complexed to a membrane structure that has been physically or chemically disrupted, e.g., by sonication or detergent treatment. Preferably, isolated phycobilisomes are stabilized, so that the phycobilisome resists dissociation in solutions of low ionic strength.
xe2x80x9cHomogeneous preparations of isolated, soluble, stabilized phycobilisomesxe2x80x9d according to this invention demonstrate homogeneity by lack of settling within a 24-hour incubation at 1xc3x97g. Solubility can be assessed by centrifugation. xe2x80x9cSoluble phycobilisome preparationxe2x80x9d means that upon centrifugation at 1,000xc3x97g for 5 minutes, greater than 55% of the phycobilisomes remain in the supernatant. It is desirable that greater than 65%, 75%, 85%, and even 90% of the phycobilisomes remain in the supernatant after such centrifugation, and such levels are possible using the methods of the present invention.
For the purpose of this invention, a xe2x80x9cphycobilisome complexxe2x80x9d is a supramolecular species of defined composition containing at least one isolated phycobilisome as defined above, the isolated phycobilisome further being specifically bound to at least one additional component. Typically, a phycobilisome complex will include more than one additional component, and may contain more than one phycobilisome. The term phycobilisome complex includes phycobilisome complexes containing, attached to, or capable of attaching to a second photosynthetic structure (e.g., an anchor polypeptide, a reaction center, a photosystem, a light-harvesting complex, a membrane protein or a membrane lipid). Further, a molecular species, which may be a ligand, a receptor or a signal-generating species, can be conjugated to the second photosynthetic structure. For example, photosystem II may be part of a phycobilisome complex and also serve as a site for conjugation of a ligand, receptor or signal-generating molecule.
The phycobilisome complex may or may not be soluble. Stabilized phycobilisome complexes comprising thylakoid membrane-associated constituents (e.g., a reaction center, photosystem light-harvesting complex, membrane protein or membrane lipid) may be insoluble in aqueous buffers and may be solubilized by treatment with surfactants, detergents, lipids, phospholipids and other amphipathic molecules well-known in the art. A phycobilisome complex may also be immobilized to a manufactured solid support.
The term xe2x80x9cligandxe2x80x9d means any substance capable of specifically binding to a receptor. Ligands include but are not limited to agonists, antagonists, biotin (or derivatives, such as amino-biotin, imino-biotin, and diamino-biotin), haptens, antigens, carbohydrates, drugs, hormones, transmitters, cofactors, vitamins, toxins, oligonucleotides, nucleic acids, aptamers, and conjugates formed by attaching any of these molecules to a second molecule. The term xe2x80x9creceptorxe2x80x9d means any substance capable of specifically binding to a ligand. Receptors include but are not limited to antibodies, antibody fragments, antibody mimetics, molecular mimics and molecular imprints, molecular recognition units, adhesion molecules, soluble receptors, avidin, streptavidin, lectins, selecting, oligonucleotides, nucleic acids, membrane receptors, cellular receptors, and drug receptors. The terms xe2x80x9cspecifically bind,xe2x80x9d xe2x80x9cspecifically boundxe2x80x9d and xe2x80x9cspecific bindingxe2x80x9d refer to the saturable, noncovalent interaction between a ligand and a receptor which is well known in the art and explicitly includes nucleic acid hybridization. xe2x80x9cHybridizationxe2x80x9d refers to specific binding between two or more nucleic acid sequences through complementary base pairing. Such binding is also referred to as Watson-Crick base pairing. For hybridization, a sufficient degree of complementarity is required to yield reversible binding between two selected nucleic acid sequences. Perfect complementarity is not required and may not be preferred for embodiments relying on reversibility, such as dissociation of a hybridized nucleic acid probe reagent by a target sequence.
xe2x80x9cSignal-generating moleculexe2x80x9d as used herein means any substance capable of generating a detectable signal or enhancing or modulating the detectability of a phycobilisome or transducing a phycobilisome signal into a qualitatively or quantitatively different signal or different form of energy. xe2x80x9cEnhancing or modulating phycobilisome detectabilityxe2x80x9d means the signal-generating molecule has an effect on phycobilisome size, shape, charge, chemical composition or spectral properties or that the phycobilisome-signal-generating molecule conjugate has different spectral properties from the unconjugated phycobilisome. xe2x80x9cTransducing the phycobilisome signalxe2x80x9d means the conjugate absorbs or emits in a different region of the electromagnetic spectrum from the unconjugated phycobilisome or the conjugate manifests an energy or function different from the unconjugated phycobilisome, such as an electric or chemical potential or catalytic activity or thermal gradient or mechanical force. Signal-generating molecules as contemplated herein include, but are not limited to, signal-generating systems comprising phycobiliproteins, dye molecules, colloids, fluorophores and other photoactive molecules, enzymes, coenzymes, cofactors, catalytic antibodies, ribozymes, and other catalytic molecules, molecular mimics, luminescent compounds, oxidizing and reducing compounds and other electroactive molecules, photosystem molecules and reaction centers not attached to phycobilisomes in nature such as artificial reaction centers, optionally including organizational, scaffold and coupling molecules used to capture energy in artificial photosynthesis, and even other phycobilisomes.
xe2x80x9cMolecular mimicsxe2x80x9d and xe2x80x9cmimeticsxe2x80x9d are synthetic molecules or groups of molecules designed or selected to perform an equivalent or similar function to that of a naturally occurring or biological molecule or group of molecules. xe2x80x9cArtificial photosynthesisxe2x80x9d refers to synthetic energy conversion systems that mimic the natural process of photosynthesis. xe2x80x9cArtificial reaction centerxe2x80x9d means a molecule or group of molecules capable of existing in a light-induced charge-separated state, thereby mimicking the function of a reaction center. Examples of artificial reaction centers are well-known in the art (Gust, et al. (1993), xe2x80x9cMolecular mimicry of photosynthetic energy and electron trasfer,xe2x80x9d Accounts of Chemical Research, 26:198-205; Gust, et al. (1994), xe2x80x9cPhotosynthesis mimics as molecular electronic devices,xe2x80x9d IEEE. Eng. Med. Biol., 13:58-66, and references therein). xe2x80x9cReaction centerxe2x80x9d means a natural photosynthetic molecule or group of molecules in which photoinitiated electron transfer culminates in a relatively long-lived, charge-separated state. The term xe2x80x9cphotosystemxe2x80x9d as used herein means a photosynthetic molecule or group of molecules that serves as a functionally coupled energy transfer acceptor from a reaction center, for example, photosystem I or photosystem II.
xe2x80x9cLight energyxe2x80x9d as defined herein is a discrete energy packet that was originally resident in a photon; light energy as contemplated herein may be transformed into other energy forms. Typically, the photon (and its light energy) will be absorbed by a pigmented substance. The light energy from the photon may be subsequently transferred from the absorbing species radiatively by photon emission, or the light energy may be non-radiatively transferred to an acceptor species.
Efficient signal or energy transduction (i.e., transfer of light energy) between a phycobilisome and an attached signal-generating molecule requires functional coupling between the two species. xe2x80x9cFunctional couplingxe2x80x9d means that two processes are connected by a common intermediate or that two species or substances participate as donor and acceptor in the transfer of mass or energy, e.g., photons or electrons or chemical or mechanical or thermal energy. The term xe2x80x9cfunctionally coupledxe2x80x9d means that a first species, substance or process is connected to a second species, substance or process by a common intermediate or by transfer of a photon, electron, property, activity, mass or energy from a donor to an acceptor. Such coupling is well known in the art (Cubicciotti, R. (1993) DNA chips. xe2x80x9cMedical and Healthcare Marketplace Guide.xe2x80x9d MLR Biomedical Information Services, 9th Edition, pp. 113-115; Cubicciotti, R. S. (1995) xe2x80x9cNucleotide-directed assembly of bimolecular and multimolecular drugs and devices.xe2x80x9d WIPO International Publication No. WO 95/16788, p. 24; Gust et al. (1993), Sheeler, P. and Bianchi, D E (1983) xe2x80x9cCell Biology: Structure, Biochemistry, and Functionxe2x80x9d, p. 203, John Wiley and Sons, Inc., New York; Saier, H S Jr. (1987), xe2x80x9cEnzymes in Metabolic Pathways: A comparative Study of Mechanism, Structure, Evolution, and Controlxe2x80x9d, pp. 48-59 and 132-136, Harper and Raw Publishers, New York; Aidley D. J. (1989), The Physiology of Excitable Cells, Third Edition, p. 320, Cambridge University Press, Cambridge; Bray, H G and White, K (1957); Kinetics and Thermodynamics in Biochemistry, p. 135, Academic Press, New York; and Guyton, A C (1971) Textbook of Medical Physiology, Fourth Edition, p. 786, W. B. Saunders Company, Philadelphia). Examples of such coupling are described by Cubicciotti (1995) to include, for example:
. . . coupling proteins to selectively or actively transport ions and metabolites; coupling cytochromes to transduce chemical energy by means of electron transfer-dependent oxidation-reduction reactions; coupling redox mediators such as ubiquinones, ferricinium salts, rubidium, viologens, tetrathiofulvalene, tetracyanoquinidodimethane, N-methylphenazinium, benzoquinone or conducting polymers or organic conducting salts to transfer electrons between electroactive molecules such as redox enzymes and electrodes in bioelectronic and optoelectronic devices such as biosensors and biochips; coupling photoactive compounds such as fluorophores with other photoactive compounds or with redox proteins or enzymes for energy transfer devices and artificial photosynthetic systems; and coupling pro-drugs for staged-delivery or triggered activation.
Phycobilisomes are said to be functionally coupled to a second molecular species or a device (e.g., a transducer) when a photon, electron, property, activity, mass or energy of a first molecule, complex or device comprising the phycobilisome is transferred to or from a second molecule, complex or device. For certain functionally coupled conjugate and biotransducer embodiments, particularly those involving phycobilbsomes, artificial reaction centers and electronic transducers, electronic coupling is preferred. xe2x80x9cElectronic couplingxe2x80x9d as used herein includes single-electron transfer and coupling mediated by direct, through-space overlap of the relevant orbitals of the donor(s) and acceptor and by through-bond superexchange(s) and may occur by single-step or multistep processes within a molecule or between molecules positioned by covalent bonding or noncovalent interaction(s).
Based on the foregoing definitions, it will be apparent to one of skill in the art that a ligand, receptor or signal-generating molecule attached to a phycobilisome can further be functionally coupled to the phycobilisome, and that functional coupling includes the exchange or transfer of mass or energy between a phycobilisome and an attached species. Functional coupling does not require direct attachment of a signal-generating molecule to a phycobilisome. A phycobilisome can be functionally coupled to a second molecule or complex or device or process indirectly, e.g., through a specific binding reaction between an attached ligand or receptor and a specific binding partner comprising a signal-generating molecule.
The term xe2x80x9cmanufactured solid supportxe2x80x9d as used herein means any structure, device, matrix or membrane which is not the native attachment site for phycobilisomes and includes non-thylakoid biological membranes and synthetic and biomimetic membranes which may comprise peptides, proteins and/or other ligands and receptors. Non-thylakoid, synthetic and biomimetic membranes can either be used directly as solid supports, or attached to or deposited or prepared on solid supports, to facilitate self-assembly, reconstitution and/or immobilization of phycobilisomes, phycobilisome subassemblies and conjugates via covalent or non-covalent attachment. In addition, phycobilisome complexes of the invention comprising thylakoid membrane fragments or constituents (e.g., lipids, proteins and membrane receptors) may be immobilized to a manufactured solid support.
The term xe2x80x9cbiotransducerxe2x80x9d as used herein means biological or biomimetic molecule(s) immobilized at and/or functionally coupled to a transducer. xe2x80x9cBiological or biomimeticxe2x80x9d molecule(s) may be isolated from biological sources or produced synthetically or may perform a function equivalent to biological molecule(s), e.g., immunologic recognition, nucleic acid hybridization, enzymatic catalysis, photosynthesis or a component reaction of photosynthesis. xe2x80x9cPhycobilisome-based biotransducerxe2x80x9d means a biotransducer comprising a phycobilisome or phycobilisome complex, wherein the phycobilisome and transducer elements are necessary and functionally inseparable components of a product or system which performs a useful function. Where an instrument or device (e.g., a microscope, fluorometer, spectrofluorometer or Clark electrode) merely performs the function of measuring a property or activity of a phycobilisome or phycobilisome preparation (e.g., size, fluorescence, absorbance or rate of oxygen evolution), the instrument is not a phycobilisome-based biotransducer, because the phycobilisome is not a component of the measuring device. Instead, in such an instance the phycobilisome or phycobilisome preparation is the object of measurement or the sample to be measured and is both structurally and functionally unnecessary to and separable from the product that performs the measurement. By contrast, the phycobilisome of a phycobilisome-based biotransducer is operatively associated with, attached to, immobilized at, packaged with, or otherwise structurally or functionally inseparable from the transducer. A phycobilisome-based biotransducer can, of course, be a two-component (or multi-component) product or system comprising a transducer component and a disposable, replaceable, reusable or upgradeable phycobilisome-containing cartridge, module, slide, disk, film, layer, fiber, connector, attachment or part that serves as an interface between the phycobilisome and the transducer. In such a case, the phycobilisome-containing component is physically separable from the transducer component but must be inserted, attached, rejoined or replaced to form the functionally coupled two-component system capable of performing the intended function. The xe2x80x9cfunctionally coupledxe2x80x9d transducer converts an activity, energy or property of the biological or biomimetic molecule(s) (e.g., the phycobilisome(s) or phycobilisome conjugate(s)) to useful work or information or a detectable signal.
Preparation of Modified Phycobilisomes
Phycobilisome Isolation
Phycobilisomes, according to the present invention, are self-assembling complexes of phycobiliproteins and linker proteins comprising at least one rod. The phycobilisomes of the present invention may be obtained from either prokaryotic cyanobacteria (blue-green algae) or eukaryotic red algae. The algae may be wild-type, mutants, hybrids, or genetic recombinants capable of expressing phycobilisome constituents. The algae may be harvested from natural environments (the wild) or grown under artificially controlled conditions. Such artificial conditions may simulate a natural environment or they may be designed to induce chromatic adaption, for example, to modulate the composition of phycobilisomes. Artificial conditions may support either autotrophic, mixotrophic, or heterotrophic growth.
General procedures for isolation of phycobilisomes from a wide range of unicellular algae have been described (see, e.g., Gantt et al. (1979) Plant Physiol. 63:615-620). Phycobilisomes can be isolated from red algae (e.g., Porphyridium cruentum) and blue-green algae (e.g., Anabaena variabilis, Spirulina platensis) by methods modified from those of Gantt and Lipschultz (1972) J. Cell Biol. 54:313-324. Typically, algal or cyanobacterial cells grown under conditions which elicit production of the photosynthetic apparatus in the cells are lysed in a phosphate buffered detergent solution. After removing cellular debris, phycobilisomes may be isolated from the aqueous supernatant by gradient centrifugation or precipitation with high concentrations of phosphate buffer (xe2x89xa71 M) or polyols (e.g., sucrose or polyethylene glycol). Isolated phycobilisomes are redissolved in phosphate buffer (about 0.75 M). Exemplary procedures are shown below.
Exemplary Procedure 1
Isolation of Phycobilisomes from Red and Blue-green Algae by Gradient Ultracentrifugation
Freshly cultured or frozen (xe2x88x9220xc2x0 C. or xe2x88x9270xc2x0 C.) algae can be cultured autotrophically in 40-500 L stirred tanks with continuous fluorescent illumination and harvested by centrifugation. Porphyridium cruentum (P. cruentum) can be grown at 20-22xc2x0 C. in an artificial seawater medium (pH 8.0) comprising sodium salts, Tadros Metals, Instant Ocean and Dunaliella vitamins. Anabaena variabilis can be grown at 25xc2x0 C. in double-strength BG-11 medium containing sodium and potassium salts, magnesium sulfate, calcium chloride, citric acid, ferric ammonium citrate and A5 Metals (pH 7.8).
Unless otherwise specified, all preparative steps can be performed at room temperature (20-23xc2x0 C.) in 0.75 M potassium phosphate (pH 7.0-7.2) optionally containing 0.05% sodium azide (KPi buffer). Twenty-four grams (wet weight) of packed cells are resuspended in 48 ml KPi buffer. PMSF (1 mM, benzamnidine (5 mM) and DNase I (10 ul of RNase-free stock at 10 U/ul) are then added, and the suspension is passed four times in 15 ml increments through a French pressure cell (Aminco) operated at 1000-1250 p.s.i. TRITON X-100 (t-octylphenoxypolyethoxyethanol, Rohm and Haas) is added to 2% and the broken cell mixture is stirred for 20 minutes. Particulate matter is removed by centrifugation at 15,000 rpm for 45 minutes in a Sorvall RC-5B Refrigerated Superspeed Centrifuge using an SS34 rotor. The supernatant is withdrawn by syringe from underneath the floating chlorophyll fraction, and approximately 9 ml is layered on each of six buffered sucrose step gradients comprising (from bottom to top) 2 M sucrose (4 ml), 1 M sucrose (8 ml), 0.5 M sucrose (7 ml) and 0.25 M sucrose (7 ml), all in 0.75 M KPi. Gradients are centrifuged 12-18 hours at 25,000 rpm in an SW27 rotor. Following centrifiugation, green, brown, brown-red, purple-red, purple and clear layers (top to bottom) can be discerned with varying resolution. Only the purple-red (rods and phycobiliprotein aggregates) and purple (phycobilisome) bands are retained. Purple-red bands are withdrawn by suction using a pasteur pipet, pooled and stored at 2-8xc2x0 C. Stabilized and conjugated rods may be prepared from this fraction, purified by gel chromatography, and immobilized. Purple phycobilisome bands in the 1.0 M sucrose layer are withdrawn, pooled, diluted four-fold with KPi buffer and centrifuged at 15,000 rpm for 40 minutes in an SS34 rotor. Resultant supernatants are withdrawn from pelleted sediment (if any) and centrifuged at 30,000 rpm for two hours in a VTi50 rotor. Final supernatants are quickly and carefully aspirated, and phycobilisome-containing pellets are resuspended in a minimal volume of KPi buffer. Protein concentration can be determined by the method of Lowry et al. (1951 J. Biol. Chem., 193:265-275). Protein measurements are carried out with the Folin phenol reagent using bovine serum albumin as standard with suitable controls for sucrose and TRITON X-100 interference. Absorption spectra were measured with a Shimadzu Model UV-160 recording spectrophotometer. Fluorescence spectra were recorded at room temperature in a 4 ml quartz cuvette with a SPEX FLUOROMAX(trademark) (scanning excitation/emission fluorometer) coupled to a Compudyne PC.
In general, phycobilisome emission spectra can be obtained by exciting phycobilisomes using light of wavelengths within the absorption spectrum of the distal sensitizing phycobiliprotein (e.g., 545 nm for P. cruentum B-PE). Phycobilisomes can be routinely characterized by 1) peak absorption per mg protein (e.g., AU545/mg for P. cruentum), 2) fluorescence signal per defined concentration (e.g., cps at Emax for intact phycobilisomes at 10 ng/ml), and 3) one or more fluorescence ratios reflecting the efficiency of inter-phycobiliprotein energy transfer (e.g., 666/573 nm emission for P. cruentum as an index of APC/B-PE coupling). Up to 24 grams wet weight of biomass can be conveniently handled using six 35 ml centrifuge tubes in an SW27 rotor for the final sucrose gradient ultracentrifugation step. Phycobilisome recovery is on the order of 0.1-1.0% of initial biomass.
Exemplary Procedure 2
Large-scale Isolation of Phycobilisomes without Gradient Ultracentrifugation
The convenience, scale and cost-effectiveness of phycobilisome isolation by conventional methods (e.g., Gantt and Lipschultz (1972) supra, Gantt et al. (1979) supra) are severely limited by the need for gradient ultracentrifugation. To enable scalable and economical production of phycobilisomes, procedures were developed for isolating phycobilisomes from different organisms without gradient ultracentrifugation. Methods based on those for Anabaena variabilis using TRITON X-100 solubilization and PEG precipitation failed to yield intact phycobilisomes from some organisms, notably P. cruentum. An additional treatment step is required to protect P. cruentum phycobilisomes during removal of Triton X-100 and PEG. Either sucrose or formaldehyde treatment was found to be effective. Summarized below is the sucrose treatment procedure, which has been validated with modification for both rhodophytes (e.g., P. cruentum) and cyanophytes (e.g., Anabaena variabilis, Spirulina platensis). Preparative scale can be readily varied by selecting different centrifuge and rotor combinations and adjusting volumes accordingly.
Cells are suspended in 5 ml 0.75 M KPi (pH 6.8) per gram wet weight. PMSF and benzamidine are added to a final concentration of 1 mM and 5 mM, respectively, and the suspension is passed through a French pressure cell three times at 1000-1250 p.s.i. Membrane-associated phycobilisomes are solubilized by treatment with 2% TRITON X-100 in 0.75 M KPi (pH 6.8) for 20 minutes with stirring. The broken cell preparation is centrifuged at 15,000 rpm for 20 minutes in a Sorvall RC-5B Refrigerated Superspeed Centrifuge using an SS34 rotor to remove membrane fragments and particulate debris. The supernatant is collected by suction from underneath the floating chlorophyll layer. The pellet is discarded. Polyethylene glycol 8000 is added to the supernatant to a concentration of 15% (wt/vol). The mixture is stirred for one hour and centrifuged for 20 minutes at 15,000 rpm in an SS34 rotor. The supernatant is discarded. The pellet is resuspended by addition of 2 M sucrose in 0.75 M KPi with gentle vortexing to a final concentration of 1.5 M sucrose. Thirty minutes following sucrose addition, the suspension is diluted approximately 4-fold with 0.75 M KPi (pH 6.8) and centrifuged for three hours at 40,000 rpm (20xc2x0 C.) in a Beckman L8-M Ultracentrifuge using a VTi50 rotor. The supernatant is discarded. The pellet is resuspended in a minimal volume of 0.75 M KPi (pH 6.8), characterized by protein, absorption and fluorescence measurements (cf. supra) and stored either refrigerated or at ambient temperature, depending on the source of phycobilisomes.
Exemplary Procedure 3
Large-scale Preparation of Phycobilisomes from Algae
An alternative large-scale isolation procedure described by Grossman and Brand (1983, Carnegie Institution of Washington Yearbook, 82, 116-120) can also be used to prepare phycobilisomes. While this procedure has not been widely used due to the requirement for large-scale preparative centrifugation, it can be adapted to a suitable scale using appropriately sized centrifuge tubes. This procedure, called the xe2x80x9crapid pelleting methodxe2x80x9d by the authors, involves breaking the cells in 1 M phosphate buffer by passing cells through a French pressure cell. The lysate is brought to 1% TRITON X-100, incubated at room temperature for 30 min, then centrifuged at 32,000xc3x97g for 30 min. With laboratory scale preparative centrifuges (e.g., Sorval RC-5A), this is done in very small centrifuge tubes (e.g., using the SS34 rotor the tubes are about 45 mL with 8 places giving about 300 mL useful volume per centrifugation run). The pellet is resuspended in 0.6 M phosphate buffer (pH 7.5) and homogenized in a glass homogenizer. TRITON X-100 is added to 1% and incubated for 30 min at room temperature. The solution is again centrifuged at 32,000xc3x97g for 30 min. A large amount of the phycobilisome then remains in solution, so the pellet is discarded. The supernatant is diluted 10-fold with 1.0 M NaKPO4 (pH 7.5) and centrifuged at 32,000xc3x97g for 1 h to bring down the phycobilisomes. This method has been applied to various algal phycobilisomes (e.g., Anacystis nidulans, Porphyridium aerugineum, Cyanidium caldarium) from a diverse group of algae (Grossman and Brand (1983) Carnegie Institution of Washington Yearbook 82, 116-120) and can be adapted for phycobilisome preparation from many species.
Exemplary Procedure 4
Preparation of Phycobilisomes from Cyanobacteria
Another alternative isolation procedure has been described for preparation of cyanobacterial phycobilisomes (Siegelman and Kycia (1982) Plant Physiol., 70:887-897). Phycobilisomes containing phycoerythrin can be isolated in the following way. Cells are lysed in a 1.0 M potassium phosphate buffer (pH 6.8) containing 1% TRITON X-100 by stirring for 1 to 1.5 h at room temperature. The suspension is centrifuged at low speed and the supernatant discarded. The pellet is resuspended in 0.5 M potassium phosphate (pH 6.8) containing 1% TRITON X-100 and centrifuged for 5 min. The supernatant containing the phycobilisomes is removed so that the chlorophyll fraction is left in the tube. The solubilized phycobilisomes are precipitated by addition of solid potassium phosphate (at a 1:1 ratio of dibasic and monobasic forms) to a final concentration of 1.5 M at pH 6.8. This is centrifuged for 10 min and the clear supernatant removed from the soft pellet containing the phycobilisomes. The phycobilisome pellet is suspended in 0.5 M potassium phosphate (pH 6.8) with 1% TRITON X-100 and precipitated again. The twice precipitated phycobilisomes are resuspended in 1.25 M potassium phosphate (pH 6.8) and stored frozen. Phycobilisomes containing no phycoerythrin can also be isolated with minimal changes to the above procedure, the changes consisting essentially of increasing the amount of potassium phosphate used to precipitate the phycobilisomes from 1.0 M to 1.25 M in several of the steps. With other minimal modifications, this method may be applied to red algae such as P. cruentum. 
Stabilization of Phycobilisomes
In agreement with published studies (e.g., Katoh (1988) Phycobilisome stability, in Methods in Enzymology Vol. 167, pp. 313-318, Academic Press; and Gantt et al., 1979, supra), isolated phycobilisomes were shown to be unstable to decreases in protein concentration and ionic strength. Using P. cruentum phycobilisomes, for example, intra-phycobilisome energy transfer was disrupted within minutes following dilution of protein (below about 1 mg/ml) or buffer (below about 0.5 M KPi), as exhibited by concentration-dependent decreases in the ratio of 666/573 nm fluorescence emission with 545 nm excitation. Similar dissociation was observed for phycobilisomes isolated from Spirulina platensis and Anabaena variabilis based on a decrease in emission of the terminal acceptor. To enable reproducible preparation of stable phycobilisome-labeled ligands and receptors for use in conventional specific binding assay configurations, phycobilisomes are preferably stabilized against dissociation.
Stabilization methods which are embraced by the present invention include covalent as well as non-covalent means. Covalent methods include crosslinking and multi-point attachment of polymers that span at least two phycobilisome constituent proteins. Crosslinking agents may be zero-length (involving the direct attachment of two phycobilisome groups without intervening spacer atoms) or they may include spacer arms of varying length. Non-covalent stabilization may be accomplished using cosolvents, such as salts and sugars, hydrophobic or affinity-based interactions, such as with certain polymers or polyvalent receptors, entrapment or encapsulation (e.g., using gels, liposomes, or micelles), or changes in physical state, such as freezing or dehydrating. Suitable methods for stabilizing phycobilisomes include the methods discussed below.
(1) Covalent stabilization can be accomplished by intra-phycobilisome (inter-subunit) crosslinking, preferably through use of short- or zero-length bifunctional reagents well-known in the art of protein modification (e.g., Wong (1991) Chemistry of Protein Conjugation and Crosslinking, CRC Press).
(2) Covalent stabilization can also be achieved by multi-site attachment of natural or synthetic polymers such as carbohydrates, lipids, oligonucleotides, proteins, peptides, polyamino acids, random or ordered copolymers of amino acids, nucleosides, sugars or other small organic molecules. This method for covalent interconnection of phycobilisome subunits can be performed using either one-step or two-step techniques. In the preferred two-step approach, a first reactant (either the phycobilisome or the bridging polymer) is activated in step one. Following removal of excess reagent, the activated reactant is attached in step two to functional groups on the second reactant.
(3) Non-covalent stabilization can be achieved using cosolvents, detergents or other buffer additives that render phycobilisome dissociation thermodynamically unfavorable. In a particular embodiment, phycobilisomes are encapsulated by sonication to form vesicles containing the phycobilisomes in a solution which promotes non-covalent stabilization (e.g., 0.75 M phosphate buffer). Suitable materials for formation of liposomes around a stabilizing solution of phycobilisomes may be readily selected by the skilled worker (see discussion of immobilization to liposomes below). Ligands or receptors or other suitable molecular species may be introduced into the liposome membrane, as is well known in the art, thereby conferring specific binding properties on the encapsulated phycobilisomes.
(4) Non-covalent, affinity-based stabilization can also be accomplished using molecules or groups of molecules having a finite affinity for functional binding sites spanning at least two phycobilisome subunits. Molecules having suitable affinity may be selected by screening or combinatorial methods from groups such as naturally occurring, modified or synthetic antibodies or antibody fragments, oligonucleotides, peptides, proteins, lectins, carbohydrates or polymers of small organic molecules. Affinity can be determined by binding studies, but suitable molecules may be more simply identified by monitoring intra-phycobilisome energy transfer upon dilution of the phycobiliprotein or buffer concentration.
Phycobilisomes may be isolated from the producing organisms after being stabilized in situ prior to cell disruption or in membrane-bound form following cell disruption. Alternatively, phycobilisomes may be isolated intact prior to in vitro stabilization or conjugation or immobilization. In yet another mode of operation, phycobiliproteins and linker proteins can be isolated and reconstituted in vitro to form phycobilisomes, which are then stabilized as described herein.
In a preferred embodiment, phycobilisomes can be stabilized through a one-step reaction with short to medium chain-length crosslinking agents. To produce stabilized phycobilisomes that remain soluble, reagents and reaction conditions are selected to favor intra-phycobilisome crosslinking over inter-phycobilisome polymerization. The medium chain-length homobifunctional dialdehyde, glutaraldehyde (GA), and the short chain-length monoaldehyde, formaldehyde (FA), are both effective in protecting phycobilisomes from dilution-induced uncoupling of energy transfer. Maximal stabilization of phycobilisomes with GA is accompanied by partial insolubilization which is only apparent following centrifugation or prolonged storage. GA-induced insolubilization can be minimized through co-optimization of GA and phycobilisome concentrations, pH, buffer concentration, and reaction time. Alternatively, conditions can be adjusted to yield GA-stabilized phycobilisomes that remain in homogeneous suspension, but sediment completely when centrifuged at 8000 g for two minutes. The stabilizing effect of GA can be improved by sequential treatment of phycobilisomes at low GA/phycobilisome mass ratio (e.g., 0.027% GA/0.727% phycobilisomes) followed by dilution of the reaction mixture with buffered GA to increase the GA/phycobilisome ratio (e.g., to 0.10% GA/0.10% phycobilisomes). In contrast to GA treatment, maximally effective stabilization with shorter chain-length crosslinkers (e.g., FA) can be achieved without loss of soluble phycobilisomes to aggregation or precipitation.
Isolated phycobilisomes, stabilized phycobilisomes and phycobilisome conjugates prepared from different cyanobacteria and rhodophytes were exposed to a diverse assortment of substances and conditions to identify chemical and environmental factors capable of modulating either the aggregation state or spectral properties of the different phycobilisome preparations. Phycobilisomes diluted to concentrations ranging from 10 ug/ml to 10 mg/ml were subjected to varying temperatures, pressures, freeze-thaw cycles, lyophilization conditions, light sources and exposures, mechanical shaking, sonication, ultracentrifugation, ultrafiltration, dialysis, electrophoresis, pH, ionic strength, buffers, acids, bases, chaotropic agents, sugars, salts, neutral and charged polymers, copolymers, ionic and nonionic detergents, polar and nonpolar solvents, oxidizing and reducing agents, protein modifying reagents and combinations of such treatments designed to reversibly modulate or irreversibly perturb the phycobilisome aggregation state or spectral properties. For all but the most extreme interventions (e.g., denaturing conditions), phycobilisome preparations could be identified with varying tolerances to each type of treatment, suggesting the possibility of using selected or engineered phycobilisomes to sense and report conditions in a particular environment or sample. Especially noteworthy were differences in phycobilisome fluorescent properties in dry and partially hydrated states as a function of stabilization and storage conditions.